Method of calibrating a fluorescent microscope using fluorescent calibration microbeads simulating stained cells

ABSTRACT

A method of calibrating a flow cytometer or fluorescent microscope is based on a set of highly uniform microbeads associated with a fluorescent dye in such a way that the microbeads have the same excitation and emission spectral properties as the samples which are to be measured. The calibration values of the microbeads are plotted against the relative fluorescence intensity peak channel or intensity reading for each microbead in the set. From this calibration plot, the relative fluorescence intensity peak channel or intensity reading of the sample is translated into equivalent soluble fluorescent dye molecules per sample particle. The calibration values of the standard microbeads are determined against solutions of the dyes. In cases where the background scatter of the bulk microbeads suspensions is too high for a direct determination against the solutions, a different set of microbeads with low background scatter is calibrated against the dye solutions and used to make an initial calibration of a flow cytometer or fluorescent microscope, which in turn, is used to calibrate the uniform microbead standards. A novel method of making the microbead standards is also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of co-pending application Serial No. 06/805,654, filed Dec. 11, 1985 now issued as U.S. Pat. No. 4,774,189, which is in turn a continuation-in-part of co-pending application Ser. No. 06/685,464, filed Dec. 24, 1984, now issued as U.S. Pat. No. 4,767,206.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to uniform, polymeric, fluorescent microbeads, the synthesis thereof and their use in aligning and calibrating flow cytometers, fluorescent microscopes, and other instruments.

2. Background Art and Reference Description

The majority of analytical cytology is conducted using highly sophisticated instruments such as fluorescence microscopes and flow cytometers to examine, count, or otherwise analyze cells stained with fluorescent dyes. Without fluorescent calibration standards, it is not possible to make quantitative determinations about the cells with respect to their fluorescence intensities.

With fluorescence microscopes, it would be useful to have fluorescent calibrated particles which have the same size and fluorescent spectral properties as the stained cells. Such particles could be used to optimize the alignment of the microscope. If such particles had calibrated numbers of fluorescent molecules on them, they could be used as an internal quantitative standard, i.e., mixed with the cells on a slide to compare the brightness of cells and particles. Quantitative estimates of the fluorescence intensity of the cells could be made by eye, or accurate measurements could be made with photocells attached to the fluorescent microscope.

Flow cytometry is the process of analyzing and sorting cells in a flowing stream. This is accomplished by intersecting the stream with an incident light, usually a laser, and detecting the resulting scattered light and fluorescence of the individual cells as a function of the particular physical characteristics or attached fluorescent dye, respectively. In addition, electronic volume sensing has also been incorporated in some flow cytometry instruments. With this array of detectors, sub-populations of cells can be analyzed and sorted in arbitrary terms by just detecting their qualitative differences. However, without size and fluorescent standards, no quantitative information on the individual cells can be gained other than the number of them counted and their proportion relative to the rest of the sample.

To determine quantitative differences between subpopulations of cells, and moreover, to give individual populations a quantitative relevance, standards are necessary with known amounts of fluorescence to which these cell samples can be compared. In FIG. 1, a microbead containing a fluorescent dye, fluorescein isothiocynate (FITC), is shown along with a cell labeled with the same dye. If a series of such microbeads containing varying amounts of the fluorescent dye is run on a flow cytometer, the resulting distributions will be obtained, as shown in FIG. 2, indicated by "Bead 1, Bead 2, and Bead 3". Now if a cell population stained with the same dye is also run on the flow cytometer under the same conditions, then the fluorescence intensity of the cells can be quantitatively compared to those of the calibrated microbeads.

Various fluorescent particles have been used in conjunction with flow cytometry including fixed cells, pollen, fluorescent microbeads, and stained nuclei. However, their use has been limited for the most part to instrument alignment and size calibration. Quantitation of fluorescence intensity of cell samples has been hampered by not having a highly uniform, stable particle which has the same excitation and emission spectra as the cells being measured. Those particles which contain the proper dyes, e.g., the fluorescein stained nuclei (marketed as Fluorotrol-GF by Ortho Diagnostic Systems, Inc.) are not stable over long periods of time and those which are considered stable, e.g., the microbeads, have not contained the same dyes which stain the cells. Highly uniform fluorescent microbeads have been available from various sources for a number of years. However, none of these beads have been suitable as quantitative standards for flow cytometry instruments because (1) many of these fluorescent microbeads are smaller than the cells to be analyzed and (2) the fluorescent dyes which have been incorporated into the small microbeads are different from those attached to the cells and (3) beads which are the preferable size (about 4-10 microns in diameter) have been expensive to produce and difficult to obtain because the techniques used in their production give particles of wide variability in size, shape, and other physical properties.

Attempts have been made to cross-calibrate microbeads containing one dye against solutions or cells containing a different dye, e.g., cumerin containing microbeads against fluorescein solutions. However, such a calibration is only good for the one excitation and emission filter system. Use of slightly different filter systems, which may occur with instruments from different manufacturers, can significantly alter the quantitative results. As recognized in parent application Serial No. 685,464, the key to having a useful fluorescent standard which can be used on any instrument or filter system is for the microbead to have the same excitation and emission spectra as the sample. A more subtle point recognized by the invention is that the environment of the dye molecules can have a large effect on the fluorescence spectra. This is demonstrated in FIG. 3, where the emission intensity of a cell labeled with fluorescein is compared to that of microbeads with fluorescein on the surface and fluorescein within the body of the hydrophobic microbead. The surface fluorescenated microbead has the fluorescein in contact with the aqueous medium, and has the same emission properties as a function of wavelength as does the fluorescein labeled cell suspended in an aqueous medium, whereas, the microbead with the fluorescein within the hydrophobic bead body has a very different response because both the excitation and emission spectra have shifted and broadened as a function of the dye being in a hydrophobic medium and not in contact with water. The related spectra as recognized by the invention are shown in FIG. 4.

Recently, larger microbeads suitable for size calibration of biological cells have been synthesized in outer space (see NASA TM 78132 "Large-size Monodisperse Latexes as a Commercial Space Product", and Patent No. 4,247,434). In addition, U.S. Pat. No. 4,336,173 to Ugelstad discloses a method for preparing uniform microbeads of a size suitable for flow cytometry calibration in a conventional, earth-bound laboratory. However, none of these large microbeads were reported to contain a fluorescent dye. Moreover, synthesis as described in Patent 4,336,173 was found to be hampered by agglomeration and high doublet formation. Even with the suggested polymeric stabilizers, the yields of mono-dispersed microbeads were lower than acceptable. The present invention has for its object, among others, the accomplishment of this task.

In the Ugelstad method, a dispersion of small, uniform, seed particles produced by conventional means is contacted with a first swelling agent which is absorbed into the particles to cause some swelling of the particles. Subsequently, a second swelling agent, usually a polymerizable monomer, is likewise absorbed into the particles. Upon internal polymerization of this monomer, the particle undergoes additional swelling. The desired particle size is reached by absorbing pre-calculated amounts of the first and second substances into the microparticle.

Ugelstad teaches that, when using an oil soluble polymerization initiator which is somewhat soluble in water, the initiator may be added after the monomer has diffused into the particles or it may be dissolved in the monomer before the latter diffuses into the particles (see Ugelstad; at column 6, line 65 to column 7, line 2). Ugelstad further teaches that, when using oil soluble initiators which are less water soluble, it will be necessary to add the initiator together with the first swelling agent (see column 7, lines 2-5). When manufacturing beads, however, adding the initiator with the first swelling agent is disadvantageous because the initiator is unstable and swelled beads containing it must be handled carefully and used quickly. By not including the initiator in the first swelling agent the manufacturer can make a large stable batch of swelled seed beads which can be stored for years, giving the manufacturer the ability to choose the initiator and second swelling agent flexibly while using the same swelled seeds, and enabling the manufacturer to get more reproducible batches of finished polymerized beads by allowing him to draw from a uniform stock solution of swelled beads. At the same time, however, it is desirable to use less water soluble initiators while manufacturing beads, as such initiators are less likely to cause agglutination among the beads and decrease the yield of monodisperse beads. In other words, the prior art teaches that, in order to get good yields of monodisperse beads, an initiator having a very low solubility in water should be used. However, the prior art further teaches that, when such an initiator is used, it should be added with the first swelling agent, and this requirement deprives the manufacturer of the advantages set forth above.

It is therefore an object of this invention to provide a method of manufacturing relatively large polymeric microbeads so that there is a very high yield of monodisperse beads without requiring the manufacturer to add the initiator to the seed beads at the same time the first swelling agent is added.

It is another object of the invention to provide a method of establishing a standard reference for calibrating a fluorescent microscope, and calibrating a fluorescent microscope for measuring cells and other particle samples. Other objects will appear as the description proceeds.

Disclosure of Invention

The present invention is based on the synthesis and calibration of a set of high-uniform, polymeric microbead standards. The microbead standards are used to align and calibrate fluorescent microscopes and flow cytometers over the size and fluorescence range of stained cells. The fluorescent microbeads of the invention exhibit excitation and emission spectra equivalent to those of the samples being measured. The calibration of fluorescence intensity of the microbeads is in terms of number of equivalent soluble fluorescent molecules per microbead.

Description of Drawings

FIG. 1 schematically illustrates a microbead containing a fluorescent dye compared with a cell labelled with the same dye.

FIG. 2 illustrates the fluorescent intensity distribution of several microbeads run on a flow cytometer.

FIG. 3 illustrates a comparison of emission intensity of a fluorescein labelled all with microbeads having fluorescein on the surface and with other microbeads having fluorescein within the body of the microbead.

FIG. 4 compares the spectra of fluorescein loaded microbeads in an aqueous medium compared with being in a non-aqueous medium.

FIG. 5 is a block diagram illustrating the steps for synthesizing the microbeads of the invention.

FIG. 6 is a block diagram of the steps involved in calibrating the fluorescent microbeads of the invention.

FIG. 7 is a block diagram illustrating calibration of an instrument utilizing the invention microbeads.

FIG. 8 illustrates microbead surface fluorescenation.

FIG. 9 is a dot plot of a set of five calibration microbeads with varying size and fluorescence intensities.

FIG. 10 is a plot on logarithmic scale of Equivalent Soluble Fluorescent Dye Molecules versus Relative Fluorescent Channel.

FIG. 11 is a calibration plot Equivalent Soluble Fluorescent Dye Molecules versus Fluorescence Intensity, for a fluorescence microscope.

Best Mode for Carrying Out the Invention Synthesis of the Microbeads

The synthesis of large-sized microbeads (1-20 microns, preferably 3-9 microns in diameter) is accomplished according to the invention by swelling seed microbeads with two or more substances. The swelling is suchthat as prescribed by thermodynamics, the enthropy of mixing within the seed microbead allows the seed to swell many times over the amount the seed would swell if mixed with a single substance. While this general approach has been previously described in Patent No. 4,336,173, further improvements are critically needed with regard to the stability of the systems and examples described in such patent. Microbeads with less tendency to agglomerate and with less tendency to form doublets are needed. The present invention thus seeks to provide a more satisfactory stability and other improved characteristics in the microbeads.

In the present invention, high concentrations of the oil soluble initiator are dissolved in the oil soluble monomers. This solution is then homogenized with an aqueous surfactant solution prior to using it to swellthe microbeads already containing an oil soluble substance. In addition, the invention system is stabilized with alkaline halide salts, preferably potassium chloride, to keep the seeds separated during swelling and polymerization. Once swollen, the suspension is purged with nitrogen and heated to initiate polymerization. It was discovered that the high concentrations of oil soluble initiator (0.5-5%) caused such rapid polymerization within the swollen seeds, that polymerization of monomer inthe aqueous phase could not proceed far enough to cause agglutination. It was further observed that any monomer/initiator droplets which were not taken up by the seeds would polymerize individually, since they contained initiator, forming a second smaller population of microbeads and again avoiding agglutination of the other microbeads, especially since the system was stabilized by the addition of salt containing large cations, e.g., potassium. Yields of the large-sized monodispersed microbeads were above 95 percent.

More specifically, the invention method is directed to making microbeads ofhighly uniform and pre-determined size. As used herein, the term "highly uniform" in reference to microbead size of a population or sample of microbeads means that the standard deviation of the diameter for the microbeads in the population or sample is approximately 2 percent or less.The microbeads are prepared from a dispersion of polymeric microparticles, hereinafter referred to as seed particles, which are highly uniform in size but substantially smaller than the size of a cell. The dispersion of seed particles is contacted with a first swelling agent, which should be asubstance of one or more materials having a molecular weight of less than 1000 and a water solubility of less than 10⁻³ g/l. The preferred first swelling agent is 1-chlorododecane. Other first swelling agents could be selected from the group consisting of cetyl alcohol, 1-chlorononane, 1-bromododecane and 1-dodecanol. Seed beads may be formed of polyvinyl 10 chloride, polyvinyl toluene, styrene, or methylmethacrylate with polyvinyl toluene preferred.

The general guidelines provided by U.S. Pat. No. 4,336,173 are considered helpful in practicing the present invention and are incorporated herein byreference. Important distinctions between the material being incorporated herein and the method of the present invention are later indicated.

The second swelling agent of the invention will always be comprised of one or more polymerizable monomers. If the bead is to be made fluorescent or have some other material connected to its surface, it is desirable to havean ethylenically unsaturated compound having a three-membered epoxy ring asone of the monomers. Representative epoxy monomers include unsaturated alkyl glycidyl esters, unsaturated alkyl glycidyl ethers, unsaturated cycloalkyl glycidyl ethers, unsaturated alkyl-substituted phenyl glycidyl ethers, and the monoepoxide compounds of the diene type monomers.

Suitable glycidyl esters include glycidyl methacrylate, glycidyl acrylate, glycidyl esters of crotonic acid and long chain unsaturated fatty acids and the like; unsaturated alkyl glycidyl ethers include vinyl glycidyl ether, isopropenyl glycidyl ether, oleyl glycidyl ether, allyl and methallyl glycidyl ethers and the like, unsaturated cycloalkyl and phenyl glycidyl ethers include 4-vinyl cyclohexyl glycidyl ether, p-vinylbenzyl glycidyl ether, o-allyl phenylglycidyl ether, and the like; the monoepoxide compounds of the diene type monomers include butadiene monoepoxide, chloroprene monoepoxide, 3,4-epoxy-1-pentene, 4,5-epoxy-1-hexene, 3,4-epoxy-1-vinylcyclohexene and divinylbenzene monoxide and the like. The preferred monomer of the foregoing is methyl methacrylate. Second string preferred monomers comprise the group consisting of styrene, vinyl tolulene, ethyl methacrylate, ethyl acrylate and methyl acrylate.

The monomer having an epoxy ring can be used alone, but is, in the preferred embodiment, combined with a second copolymerizable monomer to form the second swelling agent. This second monomer should be copolymerizable with the other monomer being used to form the second swelling agent. Suitable monomers for this purpose include the monovinylidene carbocyclic monomers, e.g., styrene, alpha-methylstyrene, ar-(t-butyl)styrene, ar-methylstyrene, ar,ar-dimethylstyrene, ar-chlorostyrene, ar-(t-amyl)styrene, ar-bromostyrene, ar-fluorostyrene, ar-cyanostyrene, ar-methoxystyrene, ar-ethylstyrene, ar-hydroxymethylstyrene, ar-ethoxystyrene, a-chloro-ar-methylstyrene, ar,ar-dichlorostyrene, ar,ar-difluorostyrene, vinyl naphthalene, and othersuch emulsion polymerizable monomers having no more than 26 carbon atoms; esters of alpha,beta-ethylenically unsaturated carboxylic acids which polymerize to form non-film forming polymers, e.g., methyl methacrylate, chloroethyl methacrylate, n-butyl methacrylate, ethyl methacrylate, isobutyl methacrylate, isopropyl methacrylate, phenyl methacrylate, butyl chloroacrylate, cyclohexyl chloroacrylate, ethyl chloroacrylate, methyl chloroacrylate, isopropyl chloroacrylate and other such esters capable of being polymerized to form hard polymers; alpha,beta-ethylenically unsaturated esters of non-polymerizable carboxylic acids, e.g., vinyl benzoate, vinyl toluate, vinyl ar-ethylbenzoate, allyl ar-ethylbenzoate, vinyl trimethylacetate, vinyl pivalate, vinyl trichloroacetate and other such monomers wherein the unsaturated moity has from 2 to 14 carbon atoms and the acid moity has from 2 to 12 carbon atoms; alpha,beta-ethylenicallyunsaturated nitriles, e.g., such as nitriles having not more than 12 carbonatoms, other polymerizable vinyl monomers such as vinyl chloride, vinyl bromide and the like. Of the foregoing, the preferred monomer, to be used in combination with glycidyl methacrylate to form the second swelling agent, is methyl methacrylate. The copolymerization parameters r₁, r₂ should match as closely as possible and still retain the solubility considerations, i.e., the second swelling should be more soluble in water than first swelling agent but still be less than 10⁻3 molarity in water. Second string monomers other than those mentioned can include those containing a carboxyl group that can be activated by carbodiimide, e.g., methacrylic acid, a primary amine group which can be activated by aldehydes, e.g., ally amine, or an isiothiocynate group whichis self reactive at alkaline Ph.

In the present invention, high concentrations of an oil soluble initiator, 0.5 to 5% by volume, are dissolved in the second swelling agent. This solution is then homogenized with an aqueous surfactant solution prior to using it to swell the microbeads already containing a first swelling agent. Homogenization aids in rapid incorporation of the monomer/ initiator solution into the microbead seeds. It is preferred, but not essential, to add a stabilizing agent to the dispersion of swelled seed particles before the addition of the second swelling agent. A preferred stabilizing agent is a salt such as a solution of an alkali metal halide in an aqueous solution of the emulsifying agent used for the swelling agent. Among the salts which may be used are the chlorides, bromides and iodides of potassium and cesium, with potassium chloride being preferred. The amount of halide salts which may be used is from about 0.001M-0.1M. Alkali metals forming hydrated ions smaller than potassium, such as sodiumand lithium, are less effective as stabilizing agents.

Once swollen, the suspension is purged with nitrogen and heated to initiatepolymerization. It was discovered that the high concentrations of oil soluble initiator (0.5-5%) caused such rapid polymerization within the swollen seeds, that polymerization of monomer in the aqueous phase could not proceed far enough to cause agglutination. It was further observed that any monomer/initiator droplets which were not taken up by the seeds would polymerize individually, since they contained initiator, forming a second smaller population of microbeads and again avoiding agglutination of the other microbeads, especially since the system was stabilized by theaddition of salt. Yields of the large-sized monodispersed microbeads were approximately 95 percent.

It is crucial that the initiator chosen be soluble in the second swelling agent, and that the solubility of the initiator in the second swelling agent be greater than its solubility in water. Chemicals suitable for use as swelling agents include benzoyl peroxide and dioctanlyl peroxide, with benzoyl peroxide preferred. Lauryl peroxide was tried in the method of theinvention, using methyl methacrylate and glycidyl methacrylate as the second swelling agent, but was found not as efficient. These solubility parameters insure that, prior to polymerization, the second swelling agentwill be more likely to escape from the beads than initiator. Moreover, mostof the initiator that does escape will be contained in droplets of free monomer rather than dissolved in the aqueous phase, thus resulting in decreased free chain polymerization and agglutination. As mentioned above,any droplets of monomer and initiator polymerize separately to form a smaller, easily separated, population of particles.

Microbead Fluorescenation

Although fluorescent dyes can be diffused into or copolymerized into the invention microbead, such microbeads may not be useful as fluorescent standards. This is so because incorporation of the dye into a non-aqueous environment can cause spectral shifts in the dye. Such spectral shifts would render the microbead near worthless as quantitative fluorescence standards. To be useful as a quantitative fluorescent standard, the microbeads have to have a functionality on their surface by which the fluorescent dye molecules can be attached. This arrangement maintains the fluorescent dye in the same aqueous environment as that of the cells labeled with the dye, thus retaining equivalent spectra. The preferred functional group on the surface of the large microbead is the epoxy group.It has versatility, in that it can be directly activated under mild conditions, pH 8.5-10, to covalently link with amine-containing dyes, e.g., fluorescein amine or phycoeyrthrine, or it can be linked to a spacergroup, e.g., 1,3-diaminopropane or 1,6-diaminohexame which in turn can be linked to a reactive dye, e.g., fluorescein isothiocynate (FITC), under similar mild conditions, pH 8.5-10, to ensure that the dye is surrounded by the aqueous medium. This scheme is illustrated in FIG. 8.

Calibration of the Fluorescent Microbead Standards

Some researchers have attempted to calibrate fluorescent microbeads as wellas proteins with radioactive labels in terms of absolute numbers of dye molecules. This approach results in a quagmire of correction factors involving quenching considerations and change in extinction coefficients due to the chemical conjugation of the dye. These problems are reflected by the fact that there are as of yet no NBS accepted primary fluorescent standards for quantitative intensity, let alone for those specific fluorescent dyes of interest in flow cytometry.

Although the idea of quantitation in terms of absolute numbers of moleculesof a fluorescent dye is attractive, its practicality at this time in unobtainable. Therefore, an alternative calibration system is provided by means of the present invention which relate a microbead standard back to astable and reproducible solution of primary standard which has the same excitation and emission spectra as the sample being measured. With sufficiently dilute solutions, considerations of quenching and changes of extinction coefficient may be avoided, as long as the spectra of the primary soluble standard solution, the microbead standards, and the labeled cells in the sample are the same. Thus, fluorescent intensities ofa sample may be related to a quantitative concentration of a soluble primary standard via calibrated microbeads which have the same spectra.

The fluorescenated microbeads are calibrated with such a system in terms ofEquivalent Soluble Dye Molecules per Microbead. For example, fluorescein microbeads are standardized against a primary laser grade fluorescein. Laser grade fluorescein was chosen because it is the most stable, and of the highest purity, of any of the fluorescein compounds. Also, it has excitation and emission spectra equivalent to that of FITC-labeled cells and the fluorescein microbead standards.

The fluorescent microbead standards are calibrated by determining the fluorescent intensity of standard solutions of laser grade dyes with a fluorometer and relating those fluorescence intensities to the fluorescence intensity of suspensions of the microbeads. The number of microbeads in the suspension per unit volume is determined with a Coulter Counter™ or a Hemocytometer™. Then from these data, the number of equivalent soluble dye molecules per microbead is calculated by dividing the equivalent soluble dye molecules per unit volume by the number of microbeads per that unit volume. In the actual practice of calibrating thefluorescent microbead standards, the refractive index of the blank beads (those unfluorescenated) is too high which causes high background scatter (250,000 equivalent soluble dye molecules per microbead). This makes it impossible to directly calibrate them in bulk suspension in a fluorometer.In this case, a different primary microbead whose scatter from the blank microbead is low (1000 equivalent soluble dye molecules per microbead) must be used for calibration against the dye solutions. Such primary microbeads may be synthesized from hydrophillic monomers as 2-hydroxy ethyl methacrylate, methacrylic acid acrylamide, and allyl fluorescein as described in Pat. Nos. 4,157,323; 4,285,819 and 4,326,008. These primary microbeads are then used to calibrate the flourescence channels of a flow cytometer, and in turn the original flourescent microbead standards are calibrated against the plot developed with the primary microbeads as shownin FIG. 10. These primary microbeads are themselves not as useful as the standard microbeads because they are too small (0.5-1.0 microns), and are not very uniform.

With specific reference to fluorescence microscopes, microscopes equipped for micro-fluorometry have been in use for a number of years. Recently, however, with the advent of new fluorescent techniques in immunology and agrowing need to obtain quantitative data, methods for rapid and convenient standardization of the measurements taken by fluorescence microscopes, e.g., as sensed by the microscope photometer or other associated photodetector, have become an absolute necessity.

The prior practice of calibrating fluorescence microscopes has involved initial calibration of the photocell of a fluorescence microscope in absolute units of radiation, followed by conversion, via a number of calculations and correction factors, to numbers of fluorescent molecules. This correlation of photocell detection measurements to numbers of fluorescent molecules then permits quantitative characterization of cells or other particle samples stained with fluorescent dye.

The method of the present invention overcomes the deficiency of the prior art associated with the need for numerous calculations involving radiationquenching (extinction) coefficients and other correction and correlation factors, by providing a rapid and convenient method of calibrating fluorescence microscopes.

The method of the present invention may be suitably be practiced with a series of highly uniform cell-sized microbeads which carry different levels of fluorochromes, e.g., fluorescent dye, calibrated in terms of equivalent soluble fluorescent molecules. These microbeads have the same excitation-emission spectra as the sample(s) to be measured. Such microbeads may, if desired, be immobilized and mounted on glass slides in such a way as to preserve the fluorescent spectra of the fluorochromes as if they were in aqueous solution, as more fully disclosed and claimed in my U.S. Pat. No. 4,689,307, issued Aug. 21, 1987 and entitled "Fluorescence Microscopy Sample Mounting Method and Structure."

The general method of the present invention may be employed for calibratinga fluorescence microscope comprising an excitation source, a photodetector,e.g., comprising a photocell and a photomultiplier tube (PMT), and, optionally, a shutter or other suitable means for controlling the exposureof the excitation source to the calibration microbeads and, subsequent to calibration, to the samples to be measured.

In the method of the invention, calibration microbeads are prepared with a fluorescent die associated therewith in a manner such that the microbeads have the same excitation and emission spectra as the cell or particle samples which are to be quantitatively measured on the fluorescence microscope.

Intensity measurements then are made for each of the calibration microbeadsby placing same on a suitable support such as a microscope slide on the stage of the microscope, focusing on a selected microbead and positioning the microscope apperture over the selected microbead under bright field conditions. The bright field light source then is turned off. Next, with aphotocell, camera, or other photometric detector associated with the fluorescent microscope, an intensity "reading" is made by actuating the excitation source, and contemporaneously (preferably immediately to avoid photo-bleaching) sensing the fluorescence intensity of the microbead sample. This fluorescence intensity determination may be carried out several times on a specific microbead sample to obtain the desired level of accuracy.

One may initially make a "zero" reading (on back ground) with non-fluorescent (blank) microbeads on the stage of the microscope, by focusing on individual microbeads, and setting the photometer to zero.

Next, one selects a microbead sample with the highest fluorescence intensity and sets the photometer to an appropriate level, e.g., by adjusting the PMT settings, to yield a suitable range of readings for the subsequently measured intensity values of microbead samples having lower fluorescence intensity than the "brightest" sample.

The foregoing procedure is then repeated for each microbead sample, utilizing several microbead populations with varying levels of fluorescence intensity, to obtain a series of intensity values for the fluorescence microscope, utilizing the respective microbead samples.

Along with the foregoing method steps in which intensity values are determined for the respective population groups of microbeads used as microbead samples, the number of equivalent soluble fluorescent dye molecules per bead in the respective highly uniform microbead samples is determined.

For each population group of highly uniform microbeads, i.e., each microbead sample, the number of equivalent soluble fluorescent dye molecules which is necessary to give rise to the same level of fluorescence intensity as the microbeads, is determined by the following steps. First, the fluorescence intensity of a suspension of the microbeadsis determined with a fluorometer as compared to solutions of the free fluorescent dye, to determine the number of equivalent soluble dye molecules per unit volume of the suspension of microbeads. Next, the number of microbeads per unit volume in the aforementioned suspension is determined, and then the number of equivalent soluble dye molecules per unit volume of the suspension of microbeads is divided by the number of microbeads per unit volume of the suspension. This yields the number of equivalent soluble fluorescent dye molecules per microbead in the specificmicrobead sample.

When the number of equivalent soluble fluorescent dye molecules per microbead is thus determined for the respective microbead samples, the fluorescence intensity values determined on the fluorescence microscope aspreviously described are utilized to construct a calibration plot of the number of equivalent soluble fluorescent dye molecules, as a function of the fluorescence intensity measured on the fluorescence microscope for therespective microbead samples.

A calibration plot of such type is shown in FIG. 11 hereof, wherein the number of equivalent soluble fluorescent dye molecules is plotted as a function of fluorescence intensity.

The respective data points on the calibration curve, viz., points A, B, C, and D, represent the empirical values determined for each of four microbead samples having different diameter and fluorescence intensity characteristics. Thus, each of the four successive samples represented by data points A, B, C, and D comprise a population of highly uniform microbeads, but the size and fluorescence intensity of the microbeads, andthe number of equivalent soluble fluorescent dye molecules associated therewith, vary from sample to sample.

As previously indicated, in some instances the background scatter of the microbeads may be too high for direct calibration thereof, and in such instances a second type of microbead, of hydrophilic character having low background scatter, is usefully employed, to carry out the method of calibrating the fluorescence microscope as described below.

The hydrophobic and hydrophilic microbeads are first prepared with fluorescent dye associated therewith in a manner such that the microbeads have the same excitation and emission spectra as the samples to be measured. The microbeads thus comprise a first batch of hydrophobic microbeads having a fluorescent dye material covalently bonded to their surfaces, and a second batch of low background scatter hydrophilic microbeads having a fluorescent dye copolymerized within the microbeads and made up of a material allowing free access of aqueous media throughoutthe hydrophilic microbeads.

Next, the low background scatter hydrophilic microbeads are calibrated against solutions of the free fluorescent dye, the dye being the same as associated with the hydrophilic and hydrophobic microbeads. This calibration is carried out by the following steps:

(1) determining that the hydrophilic microbeads in a same suspending solution have excitation and emission spectra substantially identical to solutions of the free dye;

(2) determining the fluorescent intensity of a suspension of the hydrophilic microbeads with a fluorometer as compared to solutions of the free fluorescent dye, to determine the number of equivalent soluble fluorescent dye molecules per unit volume of the hydrophilic microbead suspension;

(3) determining the number of microbeads per unit volume of the hydrophilicmicrobeads suspension; and

(4) dividing the equivalent soluble fluorescent dye molecules per unit volume of the hydrophilic microbeads suspension by the number of microbeads per unit volume of the hydrophilic microbeads suspension, to yield the number of equivalent soluble fluorescent dye molecules per hydrophilic microbead.

The hydrophilic microbeads are run on a flow cytometer to determine the fluorescence intensity channel of the flow cytometer for the hydrophilic microbead samples. A calibration plot is then constructed for the hydrophilic microbeads, of the number of equivalent soluble fluorescent dye molecules as a function of fluorescence intensity channel of the flow cytometer.

The hydrophobic microbeads then are run on the flow cytometer, to determinethe fluorescence intensity channels of the flow cytometer for the hydrophobic microbead samples. From the fluorescence intensity channels ofthe flow cytometer determined for the hydrophobic microbeads, the aforementioned calibration plot for the hydrophilic microbeads is consulted to determine, based on the fluorescence intensity channels of the flow cytometer determined for the hydrophobic microbeads, the number of equivalent soluble fluorescent dye molecules per hydrophobic microbead.

At this point, the hydrophobic microbeads have been calibrated as standard reference microbeads for the fluorescence microscope, in terms of number of equivalent soluble fluorescent dye molecules per hydrophobic microbead.

The fluorescence microscope then is calibrated with the hydrophobic microbeads, by measuring the fluorescence intensity of the hydrophobic microbeads on the fluorescence microscope. A second calibration plot then is prepared for the fluorescence microscope of the number of equivalent soluble fluorescent dye molecules as a function of fluorescence intensity measured on the fluorescence microscope for the hydrophobic microbeads.

The fluorescence microscope then is calibrated, and at the same instrument parameters as employed to establish the second calibration plot, the fluorescence intensity of the cell or particle samples may be measured on the fluorescence microscope. From such fluorescence intensity value, the number of equivalent soluble fluorescent dye molecules in the cell or particle sample is determined from the second calibration plot.

Specifically, once the fluorescence microscope calibration plot has been determined, as described above, the cell or particle sample to be quantitatively measured is placed on the fluorescence microscope. Utilizing the same PMT settings and other instrument parameters as previously employed to construct the calibration plot, the fluorescence intensity then is measured for the selected cell or particle sample, and from such intensity value, the corresponding number of equivalent soluble fluorescent dye molecules in the cell or particle sample is determined from the second calibration plot, at the measured fluorescence intensity value.

As an alternative to the calibration method set forth above, it may be useful in some instances to qualitatively compare the fluorescence intensity of cell or particle samples to the intensity of calibrated microbeads. In such qualitative method, uniform microbeads are prepared aspreviously described, with a fluorescent dye associated therewith in a manner such that the microbeads have the same size and excitation and emission spectra as the samples being measured. The microbeads are then calibrated in terms of equivalent numbers of soluble fluorescent dye molecules per microbead, as previously described. The resulting calibratedmicrobeads are mixed with the cell samples. Subsequent to mixing, the fluorescence intensity of each of the microbeads may be visually compared to the intensity of the cell or particle samples.

Alternatively, after mixing of the microbeads with the cell or particles samples, the fluorescence intensity of each of the microbeads may be measured with a photocell mounted on the microscope and such intensity quantitatively compared to the fluorescence intensity of the sample.

Synthesis of Microbeads EXAMPLE 1

One milliliter of 1-chlorododecane (CDD) was homogenized with 2.5 ml of 0.25% sodium dodecyl sulfate (SDS) in water and this was added to 5 ml of 10% suspension of 2.02 micron polyvinyl toluene microbeads in 20 ml of SDSsolution. Ten milliliters of 30% acetone in water was added to help incorporate the CDD into the microbeads. This was stirred for 12 hours before 1 ml of the suspension was added to 10 ml of distilled water and 20ml of SDS and evacuated to remove the acetone. Two hundred milligrams (4%) of benzoyl peroxide initiator was dissolved in a 5 ml solution of 95% methyl methacrylate and 5% glycidyl methacrylate before it was homogenizedwith an equal volume of 0.25% SDS solution. The ten milliliters of the homogenate was then added to the above evacuated suspension of swollen seed microbeads and the suspension was purged with nitrogen and heated to 70° C. for two hours to cause rapid polymerization of the swollen microbeads. The result was a highly uniform microbead with a diameter of 5.3 microns. The yield was 96 percent.

EXAMPLE 2

The procedure was the same as in Example 1, with the exception that 20 ml of homogenized monomers and initiator was added to the seed suspension resulting in microbeads 8.7 microns in diameter.

EXAMPLE 3

The procedure was the same as in Example 1, with the exception that 100 milligrams (2%) of benzoyl peroxide initiator was used. The results were the same as in Example 1.

EXAMPLE 4

The procedure of Example 3 was repeated except 25 mg. of benzoyl peroxide initiator (0.5%) was used. There was increased aggregation of the microbeads indicating 0.5% to be a practical lower limit of initiator concentration. There was a yield of approximately 85%.

Fluorescenation of the Microbeads EXAMPLE 5

The microbeads in Examples 1 and 2 were washed in 0.25% SDS solution and tothem was added an equal volume of 10% 1.3-diaminopropane adjusted to pH 10.0. This was stirred for 12 hours then washed in SDS solution three times and in 0.1M NaHCO₃, pH 8.5 two times. FITC was added to portions of these aminated suspensions and then they were washed four times in 0.05 M phosphate buffer pH 7.2. This resulted in green fluorescent microbeads as viewed in an epiluminescent fluorescent microscope using blue exciting light.

EXAMPLE 6

The same procedures was used as in Example 5, except Texas Red was used to replace FITC. The result was red fluorescent microbeads as viewed under the fluorescence microscope using green exciting light.

EXAMPLE 7

The microbeads obtained in Examples 1 and 2, still containing functional epoxy groups, were mixed with a solution of phycoerythrin at a pH of 9.5 for 12 hours. After washing the resulting microbead had an orange fluorescence under the fluorescence microscope.

EXAMPLE 8

The same procedure was carried out as in Example 7, except the microbeads were added to a solution of avidin. Following washing in PBS pH 7.2, the microbeads coated with avidin were exposed to a solution of biotin-phycoerythrin and the microbeads had an orange fluoresence under the fluorescence microscope.

EXAMPLE 9

The microbeads in Example 5 were fluorescenated at predetermined levels of fluorescence intensity with FITC by introducing small amounts of the dyes stepwise into the microbead suspensions and checking the microbeads fluorescence intensity with a flow cytometer. Specifically, 1 mg of FITC was dissolved in 3 ml of methanol and this was added dropwise to a suspension of microbeads from Example 1 in 0.1M NaHCO₃ at pH 8.5 while intermittently determining the fluorescence level with a flow cytometer. Only enough FITC methanol solution was added to the microbeads to reach the desired microbead fluorescence intensity. Other suspensions of the same microbeads were brought to different levels of intensity resulting in a set of microbeads of various specific predetermined fluorescence levels, i.e., 2×10⁴,4×10⁵, 9×10⁵ and 2×10⁶, which can be used to develop a calibration curve for a flow cytometer as seen in FIGS. 9 and 10.

Calibration EXAMPLE 10

Step 1

Excitation and emission spectra were taken in PBS pH 7.2 of soluble laser grade fluorescein, hydrophilic (low background scatter) 1 micron microbeads, and the microbeads from Example 6. All the excitation spectra of these samples had a peak at 293 (matched within 3 nanometers) and had equivalent shapes. They also had matching emission spectra with the peak at 518 nanometers.

Step 2

In 1000 mls. of PBS pH 7.2, 9.6 mgs of laser grade fluorescein was dissolved and further diluted 1:100 making a solution of 4.8×10 ⁻⁸ M fluorescein. The fluorescence intensity of this solution was read in a fluorometer and then ratioed against the readings of dilute microbead suspensions (1 million microbeads per ml) to determine the equivalent fluorescein molarity of the microbead suspensions. This molarity was divided by the number of microbeads per ml (1.4 million per ml) as determined with a Hemocytometer. This resulted in 7.8×10⁵ soluble fluorescein molecules per microbead.

Step 3

A set of the small calibrated hydrophilic microbeads were run on a flow cytometer and a calibration plot was made of the microbead fluorescence intensity versus the instrument fluorescent channel number. This calibration curve was then used in turn to quantitate the fluorescence intensity in terms of equivalent soluble fluorescein molecules per microbead of the set of microbeads in Example 9.

Use of the Microbeads EXAMPLE 11

A set of four fluorescent calibrated microbeads as prepared in Example 9, designated 0(blank), +1(2×10⁴), +2(9×10⁵), +3(2×10⁶), were used with a fluorescence microscope by mixing them with the stained cell samples and comparing them in intensity by eye or with a photocell to the stained cells. The designations 0, +1, +2, +3 are equivalent to the arbitrary assignments given in clinical laboratoriesand the designations blank, 2×10⁴, 9×10⁵, 2×10⁶, are in terms of equivalent soluble fluorescent moleculesper microbead. As long as the microbeads are the same size as the cells being examined, the quantitative estimate of numbers of equivalent solublefluorescent molecules per cell as determined by comparison to the microbeads will be accurate. Lymphocytes stained with FITC-Leu 1 antibody appear to have the same fluorescence intensity as the +1 microbead and taken to have 2×10⁴ equivalent soluble fluorescein molecules per cell.

EXAMPLE 12

A FACS Analyzer™ (flow cytometer) was calibrated with the microbeads in Example 9. The cells in Example 11 were run on the FACS Analyzer™ without changing any of the instrument settings. This resulted in having the cells fall in a relative intensity channel equal to 2.5×10⁴equivalent soluble fluorescein molecules per cell.

EXAMPLE 13

A Nikon Labophot fluorescence microscope comprising a mercury arc 100 watt fluorescent source and a photomuliplier tube and shutter, was equipped with a 485 (20) nanometers excitation filter, and a 520 nanometers long pass emission filter. A 10 micron objective aperture was employed on the microscope.

Four microbead populations were employed, each comprising highly uniform microbeads. The first microbead sample comprised microbeads with a diameter of 4.4 microns, the second sample comprised microbeads with a diameter of 5.4 microns, the third sample comprised microbeads with a diameter of 6.0 microns, and the fourth sample comprised microbeads with adiameter of 9.0 microns. Each of these microbead samples was calibrated in the manner previously described, to determine the number of equivalent soluble fluorescent dye molecules (molecules of fluorescein) per microbeadin each sample.

The fluorescence intensity was determined on the fluorescence microscope for the each of the microbead samples, after the microscope had been "zeroed" to a background of "blank" (dyeless) microbeads. The resulting intensity values were then employed to construct a calibration plot of thenumber of equivalent soluble fluorescent dye molecules as a function of fluorescence intensity values, as shown in FIG. 11, wherein data points A,B, C, and D represent the microbead samples of 4.4, 5.4, 6.0, and 9.0 microns diameter, respectively.

With the calibration plot thus constructed, the fluorescence microscope, atthe same PMT settings and other instrument parameters, was employed to measure the fluoresence intensity of a sample of leu 2A stained cells. This stained cell sample yielded an intensity reading of 42, from which the calibration curve yielded 1.6 x 10⁵ fluorescein molecules/cell asthe number of equivalent soluble fluorescent dye molecules per cell.

Summary

The invention in its various aspects provides a method by which a fluorescent microscope or flow cytometer may be calibrated in terms of thenumber of equivalent soluble fluorescent dye molecules per fluorescence intensity channel an intensity reading for the instrument by the use of highly uniform microbeads with a fluorescent dye associated therewith, such that the microbeads have the same excitation and emission spectra as samples being measured. The invention method is thus based on the microbeads themselves being calibrated in terms of equivalent numbers of soluble fluorescent dye molecules per microbead. Stated differently, the invention method of calibrating the microbeads is based on determining thenumber of equivalent soluble fluorescent dye molecules necessary to give rise to the same level of fluorescence intensity as the particular microbead. This is accomplished by determining the fluorescence intensity of a suspension of microbeads with a fluorometer as compared to solutions of the free fluorescent dye, and dividing by the number of microbeads in the suspension to yield the number of equivalent soluble fluorescent dye molecules per microbead. When background scatter of these microbeads is too high for direct calibration, a second type of low background scatter hydrophilic microbead is calibrated against the dye solutions and these low background scatter microbeads are then calibrated against the hydrophilic microbeads.

The calibration microbeads useful for the invention have the following properties:

(a) They are highly uniform and in the size range of the

sample cells which are being measured, 1-20u in diameter, preferably 3-9u in diameter.

(b) While primarily directed to animal cells ranging from 1-20u in diameter, those skilled in the art will immediately recognize that this invention is applicable to calibration of larger 60-100u cells, such as chloroplasts found in plants.

(c) They have associated with them a fluorescent dye that will give rise tothe same excitation and emission spectra as that of the cell sample which is being measured.

(d) They have fluorescence intensities between 10³ -10⁷ equivalent soluble molecules of fluorescent dye per microbead.

(e) They are stable with respect to size and fluorescence intensity in their suspending media, which in turn is the same as that in which the cell samples are suspended.

The calibration microbeads are composed of hydrophobic polymeric materials which have chemically functional groups on the surface of the microbeads such that a fluorescent dye may be conjugated via a stable bond, and such that this bonding will maintain the fluorescent dye in contact with the suspension medium. The hydrophobic polymeric materials are preferably a co-polymer of 95% methyl methacrylate and 5% glycidyl methacrylate, but the composition could include co-polymers in various ratios of styrene, vinyl toluene, and other acrylate and methacrylate esters with glycidyl methacrylate, allyl glycidyl ether, or other epoxy containing monomers.

The preferred method of making the calibration microbead standard is to first swell seed beads with an oil soluble compound, then following with asecond swelling with a aqueous homogenate which contains a monomer with a high concentration of oil soluble initiator (0.5-5%) dissolved in it, which will cause polymerization at such a rapid rate in the oil (monomer) phase as to minimize any polymerization in the aqueous phase, thus reducing agglomeration of the microbeads.

The preferred linkage to the microbeads of the fluorescent dye is through acovalent linkage which can be generated by first animating the surface of the microbeads with a diamine, preferably 1,3-diaminopropane or 1,6-diaminohexane, through reacting with the epoxy surface group, then reacting the aminated surface of the microbeads with a reactive fluorescent dye, such as fluorescein isothiocynate or Texas Red. However, a stable linkage can be directly formed to the microbead via the epoxy group reacting directly to a primary amine on the fluorescent dye, such asfluorescein amine or phycoerythrin.

Also to be noted is that sensitivity of the flow cytometer can be determined by using the microbead with the lowest level of fluorescence and a blank microbead, i.e. a microbead without an attached fluorescent molecule, in such a way as to determine the distance between fluorescent peaks of the two microbeads being employed for such purpose.

From the foregoing, it can be seen that the invention not only provides a unique method for calibrating a flow cytometer but also a unique synthesisof the microbead standards related thereto. 

What is claimed is:
 1. A method of calibrating a fluorescence microscope which is useable to sense fluorescence intensity, and for measuring sample fluorescence intensity of a sample suspended in a selected solvent and having a sample fluorescence due to incorporation of a sample fluorescent dye having characteristic fluorescent excitation and emission spectra and fluorescence intensity, said method comprising the steps of:(a) providing a batch of microbeads in a said selected solvent to serve as calibration microbeads, each said microbead comprising:(i) a highly-uniform diameter body, formed of a hydrophobic polymeric material; and (ii) fluorescent dye material which is the same as the sample fluorescent dye and has the same excitation and emission spectra as the sample fluorescent dye, said fluorescent dye material being covalently bonded to a surface of said body via chemical functional groups and in such manner that the fluorescent excitation and emission spectra of said covalently bonded fluorescent dye material are substantially identical to the excitation and emission spectra of the same fluorescent dye material when in a same said selected solvent forming a solution of the fluorescent dye material and also substantially identical to the excitation and emission spectra of the sample to be measured, as suspended in a same said selected solvent; (b) measuring fluorescence intensities of the fluorescent dye material in said solution comprising said dye material and a said selected solvent, wherein said solution is of known fluorescent dye material concentration; and (ii) a suspension of microbeads in a said same selected solvent, and determining a number of equivalent soluble fluorescent dye molecules per microbead for said suspension of microbeads, relative to said solution comprising said dye material and a said selected solvent; (c) measuring the fluorescence intensity of said sample on said fluorescence microscope; (d) based on the number of equivalent soluble fluorescent dye molecules per microbead determined in step (b) and fluorescence intensities for said microbeads measured on the fluorescence microscope in step (c), constructing a calibration plot for said microbeads representing equivalent soluble fluorescent dye molecules as a function of relative fluorescence intensity measurement on said fluorescence microscope; and (e) using the calibration plot and the sample fluorescence intensity to determine a number of equivalent soluble fluorescent dye molecules for the sample.
 2. A method according to claim 1, wherein said microbeads each have a diameter substantially within the range of 1 to 20 microns.
 3. A method according to claim 1, wherein said microbeads each have a diameter substantially in the range of 3-15 microns.
 4. A method of calibrating a fluorescent microscope according to claim 1, wherein the sample comprises cells.
 5. A method of calibrating a fluorescent microscope according to claim 1, wherein the sample comprises particles.
 6. A method of quantifying a sample in terms of a number of equivalent soluble fluorescent dye molecules in said sample, said sample having a fluorescent intensity and being suspended in a selected solvent, said method comprising:(a) providing a fluorescent microscope which is useable to sense fluorescence intensity, for measuring sample fluorescence intensity of a sample suspended in a selected solvent and having a sample fluorescence due to incorporation of a sample fluorescent dye having characteristic fluorescent excitation and emission spectra and fluorescence intensity; (b) calibrating said fluorescence microscope by the steps comprising:(i) providing a batch of microbeads in a said selected solvent to serve as calibration microbeads, each said microbead comprising: a highly-uniform diameter body, formed of a hydrophobic polymeric material; and fluorescent dye material which is the same as the sample fluorescent dye and has the same excitation and emission spectra as the sample fluorescent dye, said fluorescent dye material being covalently bonded to a surface of said body via chemical functional groups and in such manner that the fluorescent excitation and emission spectra of said covalently bonded fluorescent dye material are substantially identical to the excitation and emission spectra of the same fluorescent dye material when in the same said selected solvent forming a solution of the sample fluorescent dye material and also substantially identical to the excitation and emission spectra of the sample to be measured, as suspended in a same said selected solvent; (ii) measuring fluorescence intensities of the fluorescent dye material in a solution comprising said dye material and a said selected solvent, wherein said solution is of known fluorescent dye material concentration; and a suspension of microbeads in a said same selected solvent, and determing a number of equivalent soluble fluorescent dye molecules per microbead for said suspension of microbeads, relative to said solution comprising said dye material and a said selected solvent; (iii) measuring the fluorescence intensity of said sample on said fluorescence microscope; (iv) based on the number of equivalent soluble fluorescent dye molecules per microbead determined in step (ii) and fluorescence intensities for said microbeads measured on the fluorescence microscope in step (iii), constructing a calibration plot for said microbeads representing equivalent soluble fluorescent dye molecules as a function of relative fluorescence intensity measurement on said fluorescence microscope; and (v) using the calibration plot and the sample fluorescence intensity measured in step (b), substep (iii) to determine a number of equivalent soluble fluorescent dye molecules for the sample.
 7. A method of quantifying a sample according to claim 6, wherein the sample comprises cells.
 8. A method of quantifying a sample according to claim 6, wherein the sample comprises particles.
 9. A method for establishing a standard reference for calibrating a fluorescence microscope for measuring fluorescence intensity of a sample suspended in a selected solvent, and having associated excitation and emission spectra and sample fluorescence intensity, said method comprising the steps of:(a) providing in a same said selected solvent, microbeads having associated therewith a fluorescent dye in a manner such that the microbeads have a fluorescent intensity and have the same excitation and emission spectra as the sample to be measured, said microbeads having microbead surfaces and comprising a first batch of hydrophobic microbeads having a fluorescent dye material covalently bonded to said microbead surfaces, and a second batch of hydrophilic microbeads having a fluorescent dye copolymerized within the microbeads and made up of a material allowing free access of aqueous media throughout the hydrophilic microbeads; (b) determining a number of equivalent soluble fluorescent dye molecules per said hydrophilic microbead using solutions of said selected solvent containing a free fluorescent dye that is the same as the fluorescent dye associated with the microbeads, by the steps comprising:(i) determining that the hydrophilic microbeads when suspended in a solution of said selected solvent have excitation and emission spectra substantially indentical to solutions of the free fluorescent dye; (ii) determining the fluorescent intensity of a suspension of said hydrophilic microbeads with a fluorometer as compared to the fluorescent intensity of solutions of the free fluorescent dye, to determine a number of equivalent soluble fluorescent dye molecules per unit volume of the hydrophilic microbead suspension; (iii) determining a number of microbeads per unit volume of the hydrophilic microbead suspension; and (iv) dividing the number of equivalent soluble fluorescent dye molecules per unit volume of the hydrophilic microbeads suspension by the number of microbeads per unit volume of the hydrophilic microbeads suspension to yield a number of equivalent soluble fluorescent dye molecules per hydrophilic microbead; (c) determining on a flow cytometer having a plurality of fluorescence intensity channels the fluorescence intensity channel of said hydrophilic microbeads; (d) constructing a calibration plot for said hydrophilic microbeads, of the number of equivalent soluble fluorescent dye molecules as function of fluorescence intensity channel of the flow cytometer; (e) determining on said flow cytometer the fluorescence intensity channel of said hydrophobic microbeads; and (f) based on the fluorescence intensity channel determined for said hydrophobic microbeads on said flow cytometer, determining from the hydrophilic microbeads calibration plot, a number of equivalent soluble fluorescent dye molecules per hydrophobic microbead, thereby establishing said hydrophobic microbead as a standard reference.
 10. A method for establishing a standard reference for calibrating a fluorescence microscope according to claim 9, wherein the sample comprises cells.
 11. A method for establishing a standard reference for calibrating a fluorescence microscope according to claim 9, wherein the sample comprises particles.
 12. A method for calibrating a fluorescence microscope for measuring fluorescence intensity of a sample, comprising the steps of:(a) establishing a standard reference for calibrating a fluorescence microscope for measuring fluorescence intensity of a sample suspended in a selected solvent, and having associated excitation and emission spectra and sample fluorescence intensity, said method of establishing a standard reference comprising the steps of:(i) providing in the same said selected solvent, microbeads having associated therewith a fluorescent dye in a manner such that the microbeads have a fluorescent intensity and have the same excitation and emission spectra as the sample to be measured, said microbeads having microbead surfaces and comprising a first batch of hydrophobic microbeads having a fluorescent dye material covalently bonded to said microbead surfaces, and a second batch of hydrophilic microbeads having a fluorerscent dye copolymerized within the microbeads and made up of a material allowing free access of aqueous media throughout the hydrophilic microbeads; (ii) determining a number of equivalent soluble fluorescent dye molecules per said hydrophilic microbead using solutions of said selected solvent containing a free fluorescent dye that is the same as the fluorescent dye associated with the microbeads, by the steps comprising:(A) determining that the hydrophilic microbeads when suspended in a solution of said selected solvent have excitation and emission spectra substantially identical to solutions of the free fluorescent dye; (B) determining the fluorescent intensity of a suspension of said hydrophilic microbeads with a fluorometer as compared to the fluorescent intensity of solutions of the free fluorescent dye, to determine a number of equivalent soluble fluorescent dye molecules per unit volume of the hydrophilic microbead suspension; (C) determining a number of microbeads per unit volume of the hydrophilic microbead suspension; and (D) dividing the number of equivalent soluble fluorescent dye molecules per unit volume of the hydrophilic microbeads suspension by the number of microbeads per unit volume of the hydrophilic microbeads suspension to yield a number of equivalent soluble fluorescent dye molecules per hydrophilic microbead; (iii) determining on a flow cytometer having a plurality of fluorescence intensity channels the fluorescence intensity channel of said hydrophilic microbeads; (iv) constructing a calibration plot for said hydrophilic microbeads, of the number of equivalent soluble fluorescent dye molecules as function of fluorescence intensity channel of the flow cytometer; (v) determining on said flow cytometer the fluorescence intensity channel of said hydrophobic microbeads; and (vi) based on the fluorescence intensity channel determined for said hydrophobic microbeads on said flow cytometer, determining from the hydrophilic microbeads calibration plot, a number of equivalent soluble fluorescent dye molecules per hydrophobic microbeads, thereby establishing said hydrophobic microbeads as a standard reference; (b) measuring the fluorescence intensities for said standard reference hydrophobic microbeads on said fluorescence microscope; and (c) based on the number of equivalent soluble fluorescent dye molecules per hydrophobic microbead determined in step (a), and the fluorescence intensities for said hydrophobic microbeads measured on the fluorescence microscope in step (b), constructing a second calibration plot for said hydrophobic microbeads representing the number of equivalent soluble fluorescent dye molecules as a function of fluorescence intensity measured on said fluorescence microscope.
 13. A method for calibrating a fluorescence microscope for measuring fluorescence intensity of samples according to claim 12, wherein the sample comprises cells.
 14. A method for calibrating a fluorescence microscope for measuring fluorescence intensity of samples according to claim 12, wherein the sample comprises particles.
 15. A method of quantifying a sample in terms of a number of equivalent soluble fluorescent dye molecules in said sample, said sample being suspended in a selected solvent, and said method comprising:(a) providing hydrophobic microbeads associated with a fluorescent dye in a same said selected solvent, and providing said sample, said sample being stained with the same fluorescent dye as associated with said hydrophobic microbeads; (b) establishing a calibration plot utilizing a fluorescence microscope, said establishing of said calibration plot comprising the steps of:(i) providing in the same said selected solvent, microbeads having associated therewith a fluorescent dye in a manner such that the microbeads have a fluorescent intensity and have the same excitation and emission spectra as the sample to be measured, said microbeads having microbead surfaces and comprising a first batch of hydrophobic microbeads having a fluorescent dye material covalently bonded to said microbead surfaces, and a second batch of hydrophilic microbeads having a fluorerscent dye copolymerized within the microbeads and made up of a material allowing free access of aqueous media throughout the hydrophilic microbeads; (ii) determining a number of equivalent soluble fluorescent dye molecules per said hydrophilic microbead using solutions of said selected solvent containing a free fluorescent dye that is the same as the fluorescent dye associated with the microbeads, by the steps comprising:(A) determining that the hydrophilic microbeads when suspended in a solution of said selected solvent have excitation and emission spectra substantially identical to solutions of the free fluorescent dye; (B) determining the fluorescent intensity of a suspension of said hydrophilic microbeads with a fluorometer as compared to the fluorescent intensity of solutions of the free fluorescent dye, to determine a number of equivalent soluble fluorescent dye molecules per unit volume of the hydrophilic microbead suspension; (C) determining a number of microbeads per unit volume of the hydrophilic microbead suspension; and (D) dividing the number of equivalent soluble fluorescent dye molecules per unit volume of the hydrophilic microbeads suspension by the number of microbeads per unit volume of the hydrophilic microbeads suspension to yield a number of equivalent soluble fluorescent dye molecules per hydrophilic microbead; (iii) determining on a flow cytometer having a plurality of fluorescence intensity channels the fluorescence intensity channel of said hydrophilic microbeads; (iv) constructing a calibration plot for said hydrophilic microbeads, of the number of equivalent soluble fluorescent dye molecules as function of fluorescence intensity channel of the flow cytometer; (v) determining on said flow cytometer the fluorescence intensity channel of said hydrophobic microbeads; and (vi) based on the fluorescence intensity channel determined for said hydrophobic microbeads on said flow cytometer, determining from the hydrophilic microbeads calibration plot, a number of equivalent soluble fluorescent dye molecules per hydrophobic microbeads, thereby establishing said hydrophobic microbeads as a standard reference; (c) measuring the fluorescence intensity of said sample on the fluorescence microscope; and (d) from such fluorescence intensity value of the sample, determining the number of equivalent soluble fluorescent dye molecules in said sample from said second calibration plot.
 16. A method of quantifying a sample according to claim 15, wherein the sample comprises cells.
 17. A method of quantifying a sample according to claim 15, wherein the sample comprises particles. 